The overall goal of this project is to dissect the roles of the PD-1: PD-1 ligand pathway in regulating tolerance and autoimmunity. During the current funding period, we have found that PD-1 and it ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), have critical roles in regulating the balance between T cell activation and tolerance. Our studies indicate that PD-1, PD-L1 and PD-L2 limit the responses of both naove and effector self-reactive T cells. We have found that PD-L1 and PD-L2 can have overlapping roles on APC, but that PD-L1 on nonhematopoietic cells has a unique role in mediating tissue tolerance. The therapeutic potential of manipulating PD-1 and its ligands to ameliorate autoimmune disease gives impetus to further investigation of how these important immunoregulatory molecules modulate tolerance and autoimmunity. Our finding that PD- L1 on nonhematopoietic cells can inhibit T cell activation and effector function compels us to investigate how PD-L1 mediates tissue tolerance, and dissect the contributions of PD-L1 on T cells, APC, endothelial and other cell types in regulating T cell responses. Our results lead us to hypothesize that PD-1 and its ligands critically regulate the balance between pathogenic vs. protective T cell responses at multiple levels, both in the lymphoid organs and in the target tissues. To investigate this hypothesis, our specific aims are: 1) To compare the roles of PD-1 and its ligands in controlling the development and function of pathogenic myelin-reactive CD4 T cells. We will study the roles of PD-1 and its ligands in regulating the differentiation of myelin-reactive CD4 T cells to Th1, Th17 vs. FoxP3+ CD4 regulatory T cells, the effector functions of MOG-reactive Th1 vs. Th17 cells and the interplay between pathogenic Th1/Th17 effector cells and FoxP3+ regulatory T cells during the development and progression of EAE. 2) To investigate the role of the PD-1:PD-L pathway in regulating autopathogenic B cells. PD-1 and PD-L1 are expressed on B cells as well as T cells, but their roles in controlling self-reactive T vs. B cell responses are not clear. Dysregulated B cell responses may play a role in the exacerbated EAE that develops in PD-1-/- and PD-L-/- mice. We will investigate the roles of PD-1 and its ligands in the APC function of B cells, T cell: B cell collaboration and production of pathogenic antibodies by myelin-specific B cells. 3) To investigate the roles of PD-L1 and PD-L2 in tissue tolerance. We will study the roles of PD-L1 and PD-L2 in tolerance-inducing functions of dendritic cells and the role of PD-L1 on vascular endothelial cells in mediating tissue tolerance during EAE. We will use MOG 35-55/IAb tetramer together with MOG TCR transgenic, B cell IgH knockin mice and our PD-1-/-, PD-L1-/-, PD-L2-/- PD-L1/L2-/- mice to investigate how PD-1, PD-L1 and PD-L2 control tolerance and autoimmunity. These studies should further our understanding of mechanisms that control tolerance and autoimmunity, and provide insight into how PD-1 and its ligands may be effectively manipulated for therapy of autoimmune diseases.Project Narrative One of the most remarkable properties of the normal immune system is its ability to respond to and protect against a diversity of microorganisms, but not respond harmfully to cells or tissues of the human body. When regulatory mechanisms go awry, the immune system can attack the body and cause autoimmune disease. This project studies the mechanisms by which one important immunoregulatory pathway, the PD-1:PD-1 ligand pathway, regulates the responses of white blood cells, called lymphocytes, and protects against autoimmunity.